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Product FAQ

Do you offer Gateway™ vectors for expression in plants?

Answer

We do not offer any Gateway™ vectors for expression in plants.

Answer Id: E9854

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Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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Product FAQ

Can the bacluovirus infect mammalian cells? How about Drosophila cells?

Answer

Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks. Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.

Answer Id: E9433

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Product FAQ

What viral titer do you suggest having to infect my cells?

Answer

We suggest using a viral stock with a titer of >1 x 10e8 pfu/mL for expression studies.

Answer Id: E9426

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Product FAQ

How can I move my gene of interest from a Gateway™-adapted expression clone to a new Destination vector as I have lost the entry clone?

Answer

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

Answer Id: E9856

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Product FAQ

Can I scale-up the production of recombinant protein using the baculovirus expression system? If so, what methods are available?

Answer

Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae

Answer Id: E9403

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Product FAQ

Can PCR primers be tailed directly with attL sites for direct recombination into the destination vector?

Answer

No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.

Answer Id: E3212

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Product FAQ

Can I sequence a peptide that is acetylated or biotinylated?

Answer

No. These groups effectively block the N terminus.

Answer Id: E1267

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Product FAQ

Can the attB primers anneal in a non-specific manner?

Answer

No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.

Answer Id: E3199

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Product FAQ

After infecting T25 or T75 flasks with virus, when do you begin to see cell lysis and what percentage of cells will be lysed at certain time points?

Answer

This is dependent on how much virus is added. If cells are infected at an MOI of 5, usually cells are infected at 24 hours, and cells begin to lyse at around 65 hours. If less virus is used, this takes longer, and more virus takes less time.

Answer Id: E9429

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Product FAQ

Which strand is produced using M13K07 to make ssDNA from pPROEX HT or pFastBAC™ vectors?

Answer

You will get the antisense strand with pProEXHT.
You will get the + or sense strand with pFastBac I of pFastBac™ HT.

Answer Id: E3167

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Product FAQ

Can a P3 viral stock be used to generate more viruses? How about a P4 or P5 stock?

Answer

Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don’t recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.

Answer Id: E9434

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Product FAQ

What is the first step in an experiment with the Gateway™ system?

Answer

The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway™ Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway™ compatible TOPO™ Cloning vectors such as pCR™8/GW/TOPO™ and pENTR™/D-TOPO™. The final option is to use restriction enzymes to clone into a pENTR™ Dual Selection vector.

Answer Id: E3208

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Product FAQ

What primer purity should be used for adding attB sites to my PCR product?

Answer

Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway™ cloning (about 50bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.

Answer Id: E3172

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Product FAQ

What is the influence of the attB sequence on protein function, solubility, folding, and expression?

Answer

Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag) the N-terminus of an expressed protein will contain an additional 9 amino acids from the AttB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.

Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway™ cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.

Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway™ Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case the attB sites do not interfere with transcription or translation.

Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway™ Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.

Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.

Answer Id: E3223

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